( Epicurian Coli XL1-Red cells al...)Using XL1-Red Mutator Strain to Generate Esterase Variants

Epicurian Coli XL1-Red cells allow isolation of gene products with altered activity

Uwe T. Bornscheuer Markus M. Enzelberger
Institute for Technical Biochemistry, University of Stuttgart, Stuttgart, Germany

Josef Altenbuchner
Institute for Industrial Genetics, University of Stuttgart, Stuttgart, Germany

Hartmut H. Meyer
Institute for Organic Chemistry, University of Hannover, Hannover, Germany

Mutants of an esterase (PFE) from Pseudomonas fluorescens were generated using Stratagenes mutator strain, Epicurian Coli XL1-Red. One variant (A209D / L181V) stereoselectively hydrolyzed a sterically-hindered 3-hydroxy ester, which was not accepted as substrate by the wild type. After several mutation cycles, mutants were assayed by plating the esterase-producing colonies onto minimal-media agar plates containing the 3-hydroxy ethyl or glyceryl ester and indicators.

Lipases, as well as esterases, accept a wide range of non-natural esters and also exhibit high activity in organic media.^1,2 However, for sterically hindered substrates, these enzymes usually fail.

Figure 1

In previous articles, we were able to resolve aliphatic³ and arylaliphatic⁴ 3-hydroxy esters using commercial lipases or esterases with good to excellent optical purities. Still, the esters could not be resolved for the 3-hydroxy ester 1 bearing two methyl groups at carbon 4 (Figure 1). None of the 18 lipases and 2 esterases tested showed any activity. We considered several options: screen for new enzymes (tedious and time consuming), alter the reaction conditions (e.g. change the solvent system
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